CN106967675A - A kind of method of the separation and Extraction candidate stem cell from placenta - Google Patents
A kind of method of the separation and Extraction candidate stem cell from placenta Download PDFInfo
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- CN106967675A CN106967675A CN201710399878.0A CN201710399878A CN106967675A CN 106967675 A CN106967675 A CN 106967675A CN 201710399878 A CN201710399878 A CN 201710399878A CN 106967675 A CN106967675 A CN 106967675A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及细胞生物学技术领域，具体涉及一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：（1）在无菌手术室环境下采集胎盘，用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；（2）用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；（3）将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；（4）将上述收集的液体A、液体B与液体C并为混合液，离心后取细胞沉淀；（5）将细胞沉淀经过多次重悬离心处理，获得造血干细胞；（6）将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，冻存。本发明的方法能够有效提高造血干细胞分离率和细胞冻存复苏后的存活率，大大缩短的从制备到应用的周期。The present invention relates to the technical field of cell biology, in particular to a method for separating and extracting hematopoietic stem cells from placenta, comprising the following steps: (1) collecting the placenta in a sterile operating room environment, and performing pre-cleaning treatment on the placenta with a cleaning solution, Collect the processed liquid A; (2) Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the perfused liquid B; (3) Cut the placenta into pieces and digest the placental tissue with enzymatic solution , to collect the liquid C obtained after enzymatic hydrolysis; (4) combine the above collected liquid A, liquid B and liquid C into a mixed liquid, and take the cell pellet after centrifugation; (5) resuspend and centrifuge the cell pellet several times, Obtain hematopoietic stem cells; (6) Add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution, mix well, and freeze. The method of the invention can effectively improve the isolation rate of the hematopoietic stem cells and the survival rate of the cells after cryopreservation and resuscitation, and greatly shorten the period from preparation to application.
Description
技术领域technical field
本发明涉及细胞生物学技术领域，具体涉及一种从胎盘中分离提取造血干细胞的方法。The invention relates to the technical field of cell biology, in particular to a method for separating and extracting hematopoietic stem cells from placenta.
背景技术Background technique
造血干细胞，是指具有自我更新和多向分化能力的一类细胞。它的基本特性是具有自我更新能力，即经过一个细胞周期活动之后，可以产生两个与分裂前性质相同的造血干细胞，同时又具有多向分化能力，即在一定的环境条件下，造血干细胞具有向各系血细胞分化的能力。Hematopoietic stem cells refer to a class of cells with self-renewal and multidirectional differentiation capabilities. Its basic feature is self-renewal ability, that is, after a cell cycle activity, it can produce two hematopoietic stem cells with the same properties as before division, and it also has multi-directional differentiation ability, that is, under certain environmental conditions, hematopoietic stem cells have The ability to differentiate into various blood cell lines.
造血干细胞移植目前广泛应用于恶性血液病、非恶性难治性血液病、遗传性疾病和某些实体瘤治疗。造血干细胞移植是指对病人进行全身照射、化疗和免疫抑制预处理后，将正常供体或自体的造血干细胞经血管输注给病人，使重建正常的造血和免疫功能。Hematopoietic stem cell transplantation is currently widely used in the treatment of malignant blood diseases, non-malignant refractory blood diseases, genetic diseases and some solid tumors. Hematopoietic stem cell transplantation refers to the infusion of normal donor or autologous hematopoietic stem cells into the patient through blood vessels after whole-body irradiation, chemotherapy and immunosuppressive pretreatment to restore normal hematopoietic and immune functions.
一般来说，造血干细胞存在于三个部位，分别是骨髓、外周血和脐带血，根据其来源分别称之为骨髓造血干细胞、外周血造血干细胞和脐带血造血干细胞。随着医学和生物技术的发展，近年来发现胎盘里含有大量的造血干细胞，与上述三种来源的造血干细胞相比，胎盘中所含造血干细胞的数量很高，而且移植胎盘造血干细胞的配型要求不需要很严格，且移植后反应较轻且不需要采用药物。此外，作为胎盘造血干细胞来源-胎盘，来源广泛，孕妇生产后往往成为废弃物，其采集不会引起母亲和新生儿任何不适的感觉或产生任何不良的影响。诸多优点使胎盘造血干细胞有望取代骨髓造血干细胞、外周血造血干细胞和脐带血造血干细胞用于造血干细胞移植中。Generally speaking, hematopoietic stem cells exist in three parts, namely bone marrow, peripheral blood and umbilical cord blood, which are called bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cells according to their sources. With the development of medicine and biotechnology, it has been found in recent years that the placenta contains a large number of hematopoietic stem cells. Compared with the above three sources of hematopoietic stem cells, the number of hematopoietic stem cells contained in the placenta is very high, and the matching of placental hematopoietic stem cells The requirements are not very strict, and the reaction after transplantation is mild and does not require the use of drugs. In addition, placenta, as the source of placental hematopoietic stem cells, has a wide range of sources. Pregnant women often become waste after giving birth, and its collection will not cause any discomfort or adverse effects to mothers and newborns. Many advantages make placental hematopoietic stem cells promising to replace bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells and umbilical cord blood hematopoietic stem cells in hematopoietic stem cell transplantation.
人类足月胎盘存在大量的造血干细胞，比脐带血有更多的造血干细胞，而且这些胎盘造血干细胞在冷冻储存前后都可以分离出来。胎盘造血干细胞菌落形成单位(CFU)的活性是确定的，在免疫缺陷鼠中的移植实验已经证明胎盘细胞在移植中的潜力。这些结果有力地表明，人类足月胎盘有可能成为一种新型的用于移植的造血干细胞的来源。并且胎盘中有大量的造血干细胞，胎盘血干细胞是较为早期的干细胞，能在体内分化成各种细胞，胎盘血内含有丰富的各种阶段早期造血干细胞，其含量大约是脐带血的十几倍，一个胎盘中的造血干细胞可完全满足于两个成年人的需求，若能和脐带血细胞一起应用于病人，无疑大大增加了造血干细胞的含量，使这造血干细胞可完全应用于所有的适用人群。Human term placenta has a large number of hematopoietic stem cells, more than cord blood, and these placental hematopoietic stem cells can be isolated before and after cryopreservation. Placental hematopoietic stem cell colony-forming unit (CFU) activity is determined, and transplantation experiments in immunodeficient mice have demonstrated the potential of placental cells in transplantation. These results strongly suggest that human term placenta has the potential to be a novel source of hematopoietic stem cells for transplantation. And there are a large number of hematopoietic stem cells in the placenta. Placental blood stem cells are relatively early stem cells that can differentiate into various cells in the body. Placental blood contains abundant early hematopoietic stem cells of various stages, and its content is about ten times that of cord blood The hematopoietic stem cells in one placenta can fully meet the needs of two adults. If they can be applied to patients together with umbilical cord blood cells, the content of hematopoietic stem cells will undoubtedly be greatly increased, so that the hematopoietic stem cells can be completely applied to all applicable populations.
然而本领域仍然期待有新的、更有效的方法获取造血干细胞，例如期待有新的、更有效的从胎盘中分离提取造血干细胞的方法。However, the field still expects a new and more effective method for obtaining hematopoietic stem cells, for example, a new and more effective method for isolating and extracting hematopoietic stem cells from placenta.
发明内容Contents of the invention
为了克服现有技术中存在的缺点和不足，本发明的目的在于提供一种从胎盘中分离提取造血干细胞的方法，该方法节省了制备时间，能够有效提高造血干细胞分离率和细胞冻存复苏后的存活率；操作方便，效率高，大大缩短的从制备到应用的周期；且造血干细胞损伤小，数量多，纯度高，活性高，可以应用于自体或者异体的造血干细胞的移植，并且有助于受损组织的修复。In order to overcome the shortcomings and deficiencies in the prior art, the object of the present invention is to provide a method for isolating and extracting hematopoietic stem cells from the placenta. high survival rate; convenient operation, high efficiency, and greatly shortened period from preparation to application; and hematopoietic stem cells are small in damage, large in number, high in purity, and high in activity, and can be applied to autologous or allogeneic hematopoietic stem cell transplantation, and help for the repair of damaged tissue.
本发明的目的通过下述技术方案实现：一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：The object of the present invention is achieved through the following technical solutions: a method for separating and extracting hematopoietic stem cells from placenta, comprising the steps of:
（1）胎盘预处理：在无菌手术室环境下采集正常顺产或剖腹产者的健康胎盘，对采集的胎盘进行病原微生物检测，然后用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；(1) Placenta pretreatment: collect healthy placentas from normal vaginal delivery or cesarean section in a sterile operating room environment, conduct pathogenic microorganism detection on the collected placenta, and then clean the placenta with cleaning solution in the early stage, and collect the treated liquid A ;
（2）胎盘灌洗：用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；(2) Placental perfusion: Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the liquid B after perfusion;
（3）胎盘酶解：将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；(3) Enzymatic hydrolysis of placenta: Cut the placenta into pieces, digest the placental tissue with enzymatic hydrolysis solution, and collect the liquid C obtained after enzymatic hydrolysis;
（4）混合离心：将上述收集的液体A、液体B与液体C并为混合液，将混合液离心后加入DEME培养液混合均匀，得到细胞悬液，将细胞悬液离心后取细胞沉淀；(4) Mixed centrifugation: Combine the liquid A, liquid B and liquid C collected above to form a mixed solution, centrifuge the mixed solution and add DEME culture medium to mix evenly to obtain a cell suspension, centrifuge the cell suspension and take the cell pellet;
（5）重悬离心：将细胞沉淀经过多次重悬离心处理，获得造血干细胞；(5) Resuspension and centrifugation: the cell pellet was resuspended and centrifuged several times to obtain hematopoietic stem cells;
（6）细胞冻存：将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，得到细胞重悬液，将细胞重悬液分装入冻存袋中，再将冻存袋装入冻存袋盒中，进行程序降温至-78～-82℃后转至-196℃液氮罐冻存。(6) Cell cryopreservation: add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution and mix evenly to obtain a cell resuspension. Divide the cell resuspension into cryopreservation bags, and then put the cryopreservation bags into freezer bags. In the storage bag box, carry out the program to cool down to -78 ~ -82 ℃, then transfer to -196 ℃ liquid nitrogen tank for freezing.
优选的，所述步骤（1）中，检测的项目包括肝炎病毒检测、艾滋病毒检测、性病检测、组织配型检测、造血干/祖细胞定性检测。Preferably, in the step (1), the detection items include hepatitis virus detection, HIV detection, STD detection, tissue matching detection, and qualitative detection of hematopoietic stem/progenitor cells.
优选的，所述步骤（1）中，清洗液由青霉素-链霉素双抗与生理盐水按质量比为1:0.8-1.2混合制得，浓度为80-120mg/L。Preferably, in the step (1), the cleaning solution is prepared by mixing penicillin-streptomycin double antibody and normal saline at a mass ratio of 1:0.8-1.2, with a concentration of 80-120 mg/L.
优选的，所述步骤（2）中，所述灌洗液由100-300mg青霉素-链霉素双抗、0.05-0.09g酚妥拉明，40-80mg赖氨酸、1-3g维生素C、1-5mg AMD3100、2支肝素钠注射液、2支硝酸甘油注射液、30-70mL质量分数为8%-12%的PBS缓冲液和1L DEME培养液制得。Preferably, in the step (2), the lavage fluid is composed of 100-300 mg penicillin-streptomycin double antibody, 0.05-0.09 g phentolamine, 40-80 mg lysine, 1-3 g vitamin C, 1-5mg AMD3100, 2 heparin sodium injections, 2 nitroglycerin injections, 30-70mL PBS buffer with a mass fraction of 8%-12%, and 1L DEME culture solution.
优选的，所述步骤（3）中，酶解液中由0.06-0.10g胶原酶Ⅷ、0.06-0.10g胰酶、1000-5000U肝素钠、0.1-0.5g酚妥拉明、4-8g维生素C和1L DEME培养液制得。Preferably, in the step (3), the enzymolysis solution consists of 0.06-0.10g collagenase VIII, 0.06-0.10g trypsin, 1000-5000U heparin sodium, 0.1-0.5g phentolamine, 4-8g vitamin C and 1L DEME culture medium.
本发明酶解液比较温和，同时使用胶原酶Ⅷ和胰酶，不仅减少对细胞的损伤，还可以在短时间内更好的消化胎盘组织，释放出组织里面的造血干细胞，提高分离得到的胎盘造血干细胞的数量和活率；酶解液中血管扩张剂酚妥拉明具有良好的血管扩张作用。The enzymolysis solution of the present invention is relatively mild, and collagenase VIII and trypsin are used at the same time, which not only reduces the damage to cells, but also can better digest placental tissue in a short time, release hematopoietic stem cells in the tissue, and improve the quality of the isolated placenta. The number and activity rate of hematopoietic stem cells; the vasodilator phentolamine in the enzymatic hydrolysis solution has a good vasodilator effect.
优选的，所述步骤（3）中，胎盘剪碎至3-6cm，酶解液室温酶解0.5-1.5h。Preferably, in the step (3), the placenta is shredded to 3-6 cm, and the enzymatic hydrolysis solution is hydrolyzed at room temperature for 0.5-1.5 h.
优选的，所述步骤（4）中，将混合液在6-10℃温度下、1200-1800r/min转速下离心10-20min，弃上清，加入DEME培养液，得到细胞悬液。Preferably, in the step (4), the mixed solution is centrifuged at 6-10°C and 1200-1800r/min for 10-20min, the supernatant is discarded, and DEME culture medium is added to obtain a cell suspension.
优选的，所述步骤（5）中，依次用红细胞裂解液重悬细胞沉淀，室温下裂解残留的红细胞10-20min，离心后弃上清；用羟乙基淀粉溶液重悬细胞沉淀，室温下静置30-50min，取上层液体离心后弃去上清；用RPMI-1640培养基重悬细胞沉淀，离心后弃上清，得到造血干细胞。Preferably, in the step (5), the cell pellet is sequentially resuspended with erythrocyte lysate, and the remaining red blood cells are lysed at room temperature for 10-20 minutes, and the supernatant is discarded after centrifugation; the cell pellet is resuspended with hydroxyethyl starch solution, and Stand still for 30-50min, take the supernatant and centrifuge, discard the supernatant; resuspend the cell pellet with RPMI-1640 medium, discard the supernatant after centrifugation, and obtain hematopoietic stem cells.
优选的，所述步骤（6）中，造血干细胞冻存液包括如下重量份的原料：Preferably, in the step (6), the hematopoietic stem cell cryopreservation solution includes the following raw materials in parts by weight:
二甲基亚砜 10-20mLDimethyl sulfoxide 10-20mL
羟乙基淀粉 6-10gHydroxyethyl starch 6-10g
细胞培养基 4-8mLCell culture medium 4-8mL
人血清白蛋白 6-10mLHuman serum albumin 6-10mL
人自体血浆 60-100mL。Human autologous plasma 60-100mL.
所述二甲基亚砜为SIGMA型号为472301的二甲基亚砜。二甲基亚砜能提高细胞膜对水的通透性，降低溶液的冰点，加上缓慢冷冻可使细胞内的水分渗出细胞外，减少细胞内冰晶的形成，从而减少由于冰晶形成造成的细胞损伤。The dimethyl sulfoxide is dimethyl sulfoxide whose SIGMA model number is 472301. Dimethyl sulfoxide can improve the permeability of the cell membrane to water and reduce the freezing point of the solution. In addition, slow freezing can make the water in the cell seep out of the cell and reduce the formation of ice crystals in the cell, thereby reducing the cell damage caused by the formation of ice crystals. damage.
羟乙基淀粉是大分子非通透性细胞保护剂，可使细胞脱水，减少细胞内冰晶形成，对细胞膜具有保护作用。Hydroxyethyl starch is a macromolecular non-permeable cell protectant, which can dehydrate cells, reduce the formation of ice crystals in cells, and have a protective effect on cell membranes.
本发明采用在造血干细胞冻存液中添加人血白蛋白作为细胞冻存稳定剂使用，有利于在冷冻复苏过程中调节渗透压，对细胞保护作用好，并且冻存液成本低，可进一步提高冻存细胞的存储稳定性，避免由于细胞存储供者血质问题导致过程储存活性不稳定或细胞复苏率下降。In the present invention, human serum albumin is added to the cryopreservation solution of hematopoietic stem cells as a cell cryopreservation stabilizer, which is beneficial to the regulation of osmotic pressure in the process of freezing and resuscitating, has a good protective effect on cells, and the cost of the cryopreservation solution is low, which can further improve The storage stability of cryopreserved cells avoids unstable storage activity or decreased cell recovery rate due to blood quality problems of cell storage donors.
本发明的造血干细胞冻存液通过采用二甲基亚砜、羟乙基淀粉及人血清白蛋白联合应用，可以有效地使干细胞冻存实现通透性保护作用，不仅降低冰点并延缓细胞冷冻过程，还增加干细胞的非通透性细胞保护作用，少细胞内冰晶形成，有效保护细胞膜，同时实现对干细胞的直接保护作用。The hematopoietic stem cell cryopreservation solution of the present invention can effectively make the stem cell cryopreservation realize permeability protection by using dimethyl sulfoxide, hydroxyethyl starch and human serum albumin in combination, not only lowering the freezing point but also delaying the cell freezing process , It also increases the non-permeable cell protection of stem cells, reduces the formation of ice crystals in cells, effectively protects cell membranes, and at the same time realizes the direct protection of stem cells.
本发明的造血干细胞冻存液通过采用上述原料，并严格控制各原料的重量配比，制得的造血干细胞冻存液能有效保护造血干细胞免受冷冻损伤，安全性高，对细胞的损伤小，可以提高造血干细胞的复苏成活率，复苏后的成活率可以达到96%以上，且复苏后细胞活率高、细胞增殖数量大、细胞不易老化，并保证了造血干细胞复苏后的生理功能和生物学特性，微生物检测指标符合要求，延长了造血干细胞的存活期。The hematopoietic stem cell cryopreservation solution of the present invention adopts the above-mentioned raw materials and strictly controls the weight ratio of each raw material, so that the hematopoietic stem cell cryopreservation solution can effectively protect hematopoietic stem cells from freezing damage, has high safety, and has little damage to cells , can improve the recovery survival rate of hematopoietic stem cells, the survival rate after recovery can reach more than 96%, and after recovery, the cell survival rate is high, the number of cell proliferation is large, the cells are not easy to age, and the physiological function and biological function of hematopoietic stem cells after recovery are guaranteed. The biological characteristics, microbial detection indicators meet the requirements, and prolong the survival period of hematopoietic stem cells.
优选的，所述细胞培养基为X-VIVO15培养基、AIM-V培养基、DMEM/F12培养基、RPMI-1640培养基、GT-T551培养基、GT-T561培养基和Stemspan培养基中的任意一种。Preferably, the cell culture medium is X-VIVO15 medium, AIM-V medium, DMEM/F12 medium, RPMI-1640 medium, GT-T551 medium, GT-T561 medium and Stemspan medium any kind.
所述细胞培养基的种类为本领域技术人员熟知的细胞培养基即可，并无特殊的限制，本发明中优选为X-VIVO15培养基、AIM-V培养基、DMEM/F12培养基或RPMI-1640培养基。AIM-V培养基购于美国Invitrogen公司，其中主要含有L-谷氨酰胺、硫酸链霉素和硫酸庆大霉素等，用以保持造血干细胞活性，使得造血干细胞复苏后能够维持其细胞活性，并延长造血干细胞存活期。细胞培养基具有保持细胞渗透压、酸碱平衡体系和提供营养等作用。The type of the cell culture medium can be a cell culture medium well known to those skilled in the art, and there is no special limitation. In the present invention, it is preferably X-VIVO15 medium, AIM-V medium, DMEM/F12 medium or RPMI -1640 medium. AIM-V medium was purchased from Invitrogen Corporation in the United States, which mainly contains L-glutamine, streptomycin sulfate and gentamicin sulfate, etc., to maintain the activity of hematopoietic stem cells, so that the hematopoietic stem cells can maintain their cell activity after recovery. And prolong the survival of hematopoietic stem cells. Cell culture medium has the functions of maintaining cell osmotic pressure, acid-base balance system and providing nutrients.
优选的，所述人自体血浆的制备方法为：将人体外周血置于无菌离心管中，1500-2000rpm转速下离心4-8min，取上层血浆在56-60℃温度下灭活28-32min后，转移至新的离心管中，2500-3500rpm转速下离心15-25min，吸取上清，即为冻存用人自体血浆。Preferably, the preparation method of the human autologous plasma is as follows: human peripheral blood is placed in a sterile centrifuge tube, centrifuged at 1500-2000rpm for 4-8min, and the upper layer of plasma is inactivated at 56-60°C for 28-32min Afterwards, transfer to a new centrifuge tube, centrifuge at 2500-3500rpm for 15-25min, absorb the supernatant, which is human autologous plasma for cryopreservation.
人自体血浆为营养保护剂，其中包括血清蛋白、葡萄糖、无机盐离子、胰岛素、肾上腺皮质激素，类固醇激素等，不仅能够为造血干细胞提供所需的营养物质，而且血清蛋白形成了血清的粘度，可以保护细胞免受机械损伤。Human autologous plasma is a nutritional protection agent, including serum protein, glucose, inorganic salt ions, insulin, adrenocortical hormone, steroid hormones, etc., which can not only provide the required nutrients for hematopoietic stem cells, but also form the viscosity of serum, Can protect cells from mechanical damage.
本发明的细胞冻存液添加人自体血浆，人自体血浆与血清成分相似，亦含有造血干细胞生长所需的各种细胞因子及营养物质，且属于造血干细胞分离的副产物，方便获取，不存在异源性，避免了外源血清存在的安全隐患。The cell cryopreservation solution of the present invention is added with human autologous plasma, which is similar to serum components, and also contains various cytokines and nutrients required for the growth of hematopoietic stem cells, and belongs to the by-product of hematopoietic stem cell separation, which is convenient to obtain and does not exist Heterogeneity, avoiding the potential safety hazards of exogenous serum.
本发明中所述造血干细胞冻存液中含有的重组人白介素-2和人自体血浆具有显著的协同作用，可以显著提高造血干细胞的活性和复苏后的存活率。The recombinant human interleukin-2 contained in the hematopoietic stem cell cryopreservation solution of the present invention has a significant synergistic effect with human autologous plasma, and can significantly improve the activity of the hematopoietic stem cell and the survival rate after resuscitation.
本发明所述造血干细胞冻存液中采用人自体血浆作为血清替代物，不含有动物血清，因此不会引入外源蛋白，降低了动物病原污染的可能性，也不会对人体过继免疫治疗产生影响。The hematopoietic stem cell cryopreservation solution of the present invention uses human autologous plasma as a serum substitute, does not contain animal serum, and therefore does not introduce foreign proteins, reduces the possibility of animal pathogen contamination, and does not affect human adoptive immunotherapy. influences.
优选的，所述造血干细胞冻存液还包括糖类1-3g，所述糖类是由海藻糖、α-1 ,4-葡聚糖、香菇多糖和葡萄糖以重量比1:0.8-1.2：1.5-2.5：2-4组成的混合物。Preferably, the hematopoietic stem cell cryopreservation solution also includes 1-3 g of carbohydrates, which are composed of trehalose, α-1,4-glucan, lentinan and glucose in a weight ratio of 1:0.8-1.2: 1.5-2.5: a mixture of 2-4 compositions.
本发明的造血干细胞冻存液通过采用海藻糖，可以减少二甲基亚砜的使用，保证细胞在接近冰点时胞内的水分不会结晶，能有效的保护细胞，并有效降低细胞毒性。The hematopoietic stem cell cryopreservation solution of the present invention can reduce the use of dimethyl sulfoxide by using trehalose, ensure that the water in the cells will not crystallize when the cells are close to the freezing point, can effectively protect the cells, and effectively reduce cytotoxicity.
本发明添加α-1 ,4-葡聚糖能够有效的降低冻存、复苏中细胞的损伤，并能够保证复苏后细胞增殖的活力。The addition of α-1,4-glucan in the present invention can effectively reduce cell damage during cryopreservation and resuscitation, and can ensure the viability of cell proliferation after resuscitation.
本发明的造血干细胞冻存液通过采用香菇多糖和葡萄糖，可以保证复苏后细胞增殖活力不受影响。The hematopoietic stem cell cryopreservation solution of the present invention uses lentinan and glucose to ensure that the cell proliferation activity is not affected after resuscitation.
优选的，所述造血干细胞冻存液还包括非必须氨基酸0.2-0.6g和维生素C 0.1-0.5g。Preferably, the hematopoietic stem cell cryopreservation solution further includes 0.2-0.6 g of non-essential amino acids and 0.1-0.5 g of vitamin C.
所述非必须氨基酸为本领域技术人员熟知的非必须氨基酸即可，并无特殊的限制，本发明优选为多种非必需氨基酸组合而成，更优选为购买自GIBCO试剂的非必须氨基酸。The non-essential amino acids can be any non-essential amino acids well known to those skilled in the art, and there is no special limitation. The present invention is preferably a combination of various non-essential amino acids, more preferably non-essential amino acids purchased from GIBCO reagents.
本发明的造血干细胞冻存液通过采用非必须氨基酸和维生素C，可以保证复苏后细胞增殖活力不受影响。The hematopoietic stem cell cryopreservation solution of the present invention can ensure that cell proliferation activity is not affected after resuscitation by using non-essential amino acids and vitamin C.
优选的，所述造血干细胞冻存液还包括丙二醇0.2-0.6mL和聚乙二醇0.4-0.8mL。Preferably, the hematopoietic stem cell cryopreservation solution further includes 0.2-0.6 mL of propylene glycol and 0.4-0.8 mL of polyethylene glycol.
本发明在造血干细胞冻存液中添加聚乙二醇和1,2-丙二醇，可以替代现用的二甲基亚砜为基础的冻存液，减少二甲基亚砜的用量，有效降低其对细胞毒性，维持细胞稳定性，提高复苏后直接应用的安全性，适合用于细胞冻存复苏后直接应用。The present invention adds polyethylene glycol and 1,2-propanediol to the cryopreservation solution of hematopoietic stem cells, which can replace the currently used dimethyl sulfoxide-based cryopreservation solution, reduce the dosage of dimethyl sulfoxide, and effectively reduce its impact on Cytotoxicity, maintain cell stability, improve the safety of direct application after resuscitation, suitable for direct application after cryopreservation and resuscitation.
优选的，所述造血干细胞冻存液还包括重组人白介素4000-8000U。所述重组人白介素可以采用注射用重组人白介素-2。Preferably, the hematopoietic stem cell cryopreservation solution further includes 4000-8000 U of recombinant human interleukin. The recombinant human interleukin can be recombinant human interleukin-2 for injection.
通常，外周血的T细胞、B细胞和NK细胞膜表面均存在白介素-2(IL-2)受体，IL-2与其受体结合后可以调节该细胞的增生，调节免疫球蛋白的生成；促进T细胞、B细胞和NK细胞的增殖、分化并能提高NK细胞的杀伤活性。然而，目前主要是将IL-2作为体外刺激培养物用于造血干细胞的扩增培养，而本发明采用将重组人白介素-2(IL-2)添加到造血干细胞冻存液中，可大幅度提高所述造血干细胞的活性稳定性，使其保持原有细胞高活性，从而使造血干细胞很好地保持了细胞复苏后的生理功能和生物学特性，可有效解决细胞复苏后直接应用和进一步扩展的关键问题。Usually, there are interleukin-2 (IL-2) receptors on the membrane surface of T cells, B cells and NK cells in peripheral blood. After IL-2 binds to its receptors, it can regulate the proliferation of the cells and regulate the production of immunoglobulins; Proliferation and differentiation of T cells, B cells and NK cells and can increase the killing activity of NK cells. However, at present, IL-2 is mainly used as an in vitro stimulating culture for the expansion and cultivation of hematopoietic stem cells, and the present invention adopts adding recombinant human interleukin-2 (IL-2) to the hematopoietic stem cell cryopreservation liquid, which can significantly Improve the stability of the activity of the hematopoietic stem cells, so that they can maintain the high activity of the original cells, so that the hematopoietic stem cells can well maintain the physiological functions and biological characteristics of the cells after recovery, which can effectively solve the problem of direct application and further expansion of the cells after recovery. key issues.
本发明添加重组人白介素-2能够有效维持细胞的活性，保证细胞复苏后正常的生物学功能。The addition of recombinant human interleukin-2 in the invention can effectively maintain the activity of cells and ensure the normal biological function of cells after recovery.
优选的，所述造血干细胞冻存液还包括白酒草皂苷R 0.3-0.7g和勃脉力A 0.8-1.2g。Preferably, the hematopoietic stem cell cryopreservation solution also includes 0.3-0.7 g of Liquor saponin R and 0.8-1.2 g of Bomaili A.
本发明的造血干细胞冻存液通过采用白酒草皂苷R与二甲基亚砜结合制备造血干细胞的冻存液，可以降低二甲基亚砜的使用量，这种冻存液对冻存细胞有较好的保护作用，冻存细胞复苏后依然保持良好的细胞活力，细胞回收率高。The cryopreservation solution for hematopoietic stem cells of the present invention can reduce the amount of dimethyl sulfoxide used by combining liquorgrass saponin R and dimethyl sulfoxide to prepare the cryopreservation solution for hematopoietic stem cells. Good protective effect, frozen cells still maintain good cell viability after recovery, and the cell recovery rate is high.
本发明的造血干细胞冻存液通过采用勃脉力A，可以维持电解质平衡，使细胞处于一种比较温和的更接近于体内环境的溶液中。The hematopoietic stem cell cryopreservation solution of the present invention can maintain the electrolyte balance by using Bomali A, so that the cells are in a milder solution that is closer to the environment in the body.
优选的，所述造血干细胞冻存液还包括青霉素-链霉素双抗溶液0.1-0.5mL和氯化钠0.4-0.8g。Preferably, the hematopoietic stem cell cryopreservation solution also includes 0.1-0.5 mL of penicillin-streptomycin double antibody solution and 0.4-0.8 g of sodium chloride.
所述青霉素和链霉素双抗溶液为GIBCO青霉素和链霉素双抗溶液GIBCO青霉素和链霉素双抗溶液每毫升包含10000单位青霉素（碱）和 10000µg链霉素（碱），利用0.85%盐液形式的青霉素G（钠盐）和硫酸链霉素，其抗菌谱包括革兰氏阳性和革兰氏阴性细菌。青霉素和链霉素双抗溶液可保证细胞储存过程中处于无菌状态，且对造血干细胞无不良影响，安全可靠。The penicillin and streptomycin double-antibody solution is GIBCO penicillin and streptomycin double-antibody solution GIBCO penicillin and streptomycin double-antibody solution contains 10,000 units of penicillin (base) and 10,000 µg of streptomycin (base) per milliliter, using 0.85% Penicillin G (sodium salt) and streptomycin sulfate in saline form have an antibacterial spectrum that includes Gram-positive and Gram-negative bacteria. The penicillin and streptomycin double antibody solution can ensure that the cells are in a sterile state during storage, and has no adverse effects on hematopoietic stem cells, which is safe and reliable.
本发明的造血干细胞冻存液通过采用氯化钠，可以维持电解质平衡，使细胞处于一种比较温和的更接近于体内环境的溶液中。The hematopoietic stem cell cryopreservation solution of the present invention can maintain the electrolyte balance by using sodium chloride, so that the cells are in a milder solution closer to the internal environment.
优选的，所述步骤（6）中，细胞重悬液的细胞密度为5×106-5×107个/mL；程序降温先以1-2℃/min的降温速度降温至-23～-27℃，再以5-7℃/min的降温速度降温至-78～-82℃。Preferably, in the step (6), the cell density of the cell resuspension is 5×10 6 -5×10 7 cells/mL; the temperature is lowered at a rate of 1-2°C/min to -23~ -27°C, then lower the temperature to -78～-82°C at a cooling rate of 5-7°C/min.
本发明的有益效果在于：本发明的方法节省了制备时间，能够有效提高造血干细胞分离率和细胞冻存复苏后的存活率；操作方便，效率高，大大缩短的从制备到应用的周期；且造血干细胞损伤小，数量多，纯度高，活性高，可以应用于自体或者异体的造血干细胞的移植，并且有助于受损组织的修复。The beneficial effects of the present invention are: the method of the present invention saves preparation time, can effectively improve the separation rate of hematopoietic stem cells and the survival rate of cells after cryopreservation and resuscitation; the operation is convenient, the efficiency is high, and the cycle from preparation to application is greatly shortened; and Hematopoietic stem cells are small in damage, large in number, high in purity, and high in activity, and can be applied to autologous or allogeneic hematopoietic stem cell transplantation, and help repair damaged tissues.
本发明的整个操作过程简单有效，实验操作时间短，显著提高分离提取效率，获得的细胞数量大，造血干细胞的含量大大提高，细胞数量较稳定；同时优化提取分离的条件，尽可能的提高造血干细胞的存活率，细胞污染率极低。The whole operation process of the present invention is simple and effective, the experimental operation time is short, the efficiency of separation and extraction is significantly improved, the number of cells obtained is large, the content of hematopoietic stem cells is greatly increased, and the number of cells is relatively stable; at the same time, the conditions for extraction and separation are optimized to improve hematopoietic stem cells as much as possible. The survival rate of stem cells and the cell contamination rate are extremely low.
具体实施方式detailed description
为了便于本领域技术人员的理解，下面结合实施例对本发明作进一步的说明，实施方式提及的内容并非对本发明的限定。In order to facilitate the understanding of those skilled in the art, the present invention will be further described below in conjunction with the examples, and the contents mentioned in the embodiments are not intended to limit the present invention.
实施例1Example 1
一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：A method for isolating and extracting hematopoietic stem cells from placenta, comprising the steps of:
（1）胎盘预处理：在无菌手术室环境下采集正常顺产或剖腹产者的健康胎盘，对采集的胎盘进行病原微生物检测，然后用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；(1) Placenta pretreatment: collect healthy placentas from normal vaginal delivery or cesarean section in a sterile operating room environment, conduct pathogenic microorganism detection on the collected placenta, and then clean the placenta with cleaning solution in the early stage, and collect the treated liquid A ;
（2）胎盘灌洗：用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；(2) Placental perfusion: Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the liquid B after perfusion;
（3）胎盘酶解：将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；(3) Enzymatic hydrolysis of placenta: Cut the placenta into pieces, digest the placental tissue with enzymatic hydrolysis solution, and collect the liquid C obtained after enzymatic hydrolysis;
（4）混合离心：将上述收集的液体A、液体B与液体C并为混合液，将混合液离心后加入DEME培养液混合均匀，得到细胞悬液，将细胞悬液离心后取细胞沉淀；(4) Mixed centrifugation: Combine the liquid A, liquid B and liquid C collected above to form a mixed solution, centrifuge the mixed solution and add DEME culture medium to mix evenly to obtain a cell suspension, centrifuge the cell suspension and take the cell pellet;
（5）重悬离心：将细胞沉淀经过多次重悬离心处理，获得造血干细胞；(5) Resuspension and centrifugation: the cell pellet was resuspended and centrifuged several times to obtain hematopoietic stem cells;
（6）细胞冻存：将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，得到细胞重悬液，将细胞重悬液分装入冻存袋中，再将冻存袋装入冻存袋盒中，进行程序降温至-78℃后转至-196℃液氮罐冻存。(6) Cell cryopreservation: add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution and mix evenly to obtain a cell resuspension. Divide the cell resuspension into cryopreservation bags, and then put the cryopreservation bags into freezer bags. In the storage bag box, the temperature was lowered to -78°C and then transferred to -196°C liquid nitrogen tank for freezing.
所述步骤（1）中，检测的项目包括肝炎病毒检测、艾滋病毒检测、性病检测、组织配型检测、造血干/祖细胞定性检测。In the step (1), the detection items include hepatitis virus detection, HIV detection, STD detection, tissue matching detection, and qualitative detection of hematopoietic stem/progenitor cells.
所述步骤（1）中，清洗液由青霉素-链霉素双抗与生理盐水按质量比为1:0.8混合制得，浓度为80mg/L。In the step (1), the cleaning solution is prepared by mixing penicillin-streptomycin double antibody and normal saline at a mass ratio of 1:0.8, with a concentration of 80 mg/L.
所述步骤（2）中，所述灌洗液由100mg青霉素-链霉素双抗、0.05g酚妥拉明，40mg赖氨酸、1g维生素C、1mg AMD3100、2支肝素钠注射液（1mL：5mg）、2支硝酸甘油注射液（2mL：12500U）、30mL质量分数为8%的PBS缓冲液和1L DEME培养液制得。In the step (2), the lavage solution is composed of 100 mg penicillin-streptomycin double antibody, 0.05 g phentolamine, 40 mg lysine, 1 g vitamin C, 1 mg AMD3100, 2 heparin sodium injections (1 mL : 5mg), 2 injections of nitroglycerin (2mL: 12500U), 30mL of 8% PBS buffer and 1L of DEME culture solution.
所述步骤（3）中，酶解液中由0.06g胶原酶Ⅷ、0.06g胰酶、1000U肝素钠、0.1g酚妥拉明、4g维生素C和1L DEME培养液制得。In the step (3), the enzymolysis solution is prepared from 0.06g collagenase VIII, 0.06g trypsin, 1000U heparin sodium, 0.1g phentolamine, 4g vitamin C and 1L DEME culture solution.
所述步骤（3）中，胎盘剪碎至3cm，酶解液室温酶解0.5h。In the step (3), the placenta is shredded to 3 cm, and the enzymatic hydrolysis solution is hydrolyzed at room temperature for 0.5 h.
所述步骤（4）中，将混合液在6℃温度下、1200r/min转速下离心20min，弃上清，加入DEME培养液，得到细胞悬液。In the step (4), the mixture was centrifuged at 6° C. and 1200 r/min for 20 minutes, the supernatant was discarded, and DEME culture medium was added to obtain a cell suspension.
所述步骤（5）中，依次用红细胞裂解液重悬细胞沉淀，室温下裂解残留的红细胞10min，离心后弃上清；用羟乙基淀粉溶液重悬细胞沉淀，室温下静置30min，取上层液体离心后弃去上清；用RPMI-1640培养基重悬细胞沉淀，离心后弃上清，得到造血干细胞。In the step (5), resuspend the cell pellet with erythrocyte lysate in turn, lyse the residual red blood cells at room temperature for 10 minutes, discard the supernatant after centrifugation; resuspend the cell pellet with hydroxyethyl starch solution, let stand at room temperature for 30 minutes, take After the supernatant was centrifuged, the supernatant was discarded; the cell pellet was resuspended in RPMI-1640 medium, and the supernatant was discarded after centrifugation to obtain hematopoietic stem cells.
所述步骤（6）中，造血干细胞冻存液包括如下重量份的原料：In the step (6), the hematopoietic stem cell cryopreservation solution includes the following raw materials in parts by weight:
二甲基亚砜 10mLDimethyl sulfoxide 10mL
羟乙基淀粉 6gHydroxyethyl starch 6g
细胞培养基 4mLCell culture medium 4mL
人血清白蛋白 6mLHuman Serum Albumin 6mL
人自体血浆 60mL。Human autologous plasma 60mL.
所述步骤（6）中，细胞重悬液的细胞密度为5×106个/mL；程序降温先以1℃/min的降温速度降温至-23℃，再以5℃/min的降温速度降温至-78℃。In the step (6), the cell density of the cell resuspension is 5×10 6 cells/mL; the programmed cooling first cools down to -23°C at a cooling rate of 1°C/min, and then cools at a cooling rate of 5°C/min Cool down to -78°C.
实施例2Example 2
一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：A method for isolating and extracting hematopoietic stem cells from placenta, comprising the steps of:
（1）胎盘预处理：在无菌手术室环境下采集正常顺产或剖腹产者的健康胎盘，对采集的胎盘进行病原微生物检测，然后用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；(1) Placenta pretreatment: collect healthy placentas from normal vaginal delivery or cesarean section in a sterile operating room environment, conduct pathogenic microorganism detection on the collected placenta, and then clean the placenta with cleaning solution in the early stage, and collect the treated liquid A ;
（2）胎盘灌洗：用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；(2) Placental perfusion: Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the liquid B after perfusion;
（3）胎盘酶解：将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；(3) Enzymatic hydrolysis of placenta: Cut the placenta into pieces, digest the placental tissue with enzymatic hydrolysis solution, and collect the liquid C obtained after enzymatic hydrolysis;
（4）混合离心：将上述收集的液体A、液体B与液体C并为混合液，将混合液离心后加入DEME培养液混合均匀，得到细胞悬液，将细胞悬液离心后取细胞沉淀；(4) Mixed centrifugation: Combine the liquid A, liquid B and liquid C collected above to form a mixed solution, centrifuge the mixed solution and add DEME culture medium to mix evenly to obtain a cell suspension, centrifuge the cell suspension and take the cell pellet;
（5）重悬离心：将细胞沉淀经过多次重悬离心处理，获得造血干细胞；(5) Resuspension and centrifugation: the cell pellet was resuspended and centrifuged several times to obtain hematopoietic stem cells;
（6）细胞冻存：将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，得到细胞重悬液，将细胞重悬液分装入冻存袋中，再将冻存袋装入冻存袋盒中，进行程序降温至-79℃后转至-196℃液氮罐冻存。(6) Cell cryopreservation: add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution and mix evenly to obtain a cell resuspension. Divide the cell resuspension into cryopreservation bags, and then put the cryopreservation bags into freezer bags. In the storage bag box, the temperature was lowered to -79°C and then transferred to -196°C liquid nitrogen tank for freezing.
所述步骤（1）中，检测的项目包括肝炎病毒检测、艾滋病毒检测、性病检测、组织配型检测、造血干/祖细胞定性检测。In the step (1), the detection items include hepatitis virus detection, HIV detection, STD detection, tissue matching detection, and qualitative detection of hematopoietic stem/progenitor cells.
所述步骤（1）中，清洗液由青霉素-链霉素双抗与生理盐水按质量比为1:0.9混合制得，浓度为90mg/L。In the step (1), the cleaning solution is prepared by mixing penicillin-streptomycin double antibody and normal saline at a mass ratio of 1:0.9, with a concentration of 90 mg/L.
所述步骤（2）中，所述灌洗液由150mg青霉素-链霉素双抗、0.06g酚妥拉明，50mg赖氨酸、1.5g维生素C、2mg AMD3100、2支肝素钠注射液（1mL：5mg）、2支硝酸甘油注射液（2mL：12500U）、40mL质量分数为9%的PBS缓冲液和1L DEME培养液制得。In the step (2), the lavage solution is composed of 150 mg penicillin-streptomycin double antibody, 0.06 g phentolamine, 50 mg lysine, 1.5 g vitamin C, 2 mg AMD3100, 2 heparin sodium injections ( 1mL: 5mg), 2 injections of nitroglycerin (2mL: 12500U), 40mL of 9% PBS buffer and 1L DEME culture solution.
所述步骤（3）中，酶解液中由0.07g胶原酶Ⅷ、0.07g胰酶、2000U肝素钠、0.2g酚妥拉明、5g维生素C和1L DEME培养液制得。In the step (3), the enzymolysis solution is prepared from 0.07g collagenase VIII, 0.07g trypsin, 2000U heparin sodium, 0.2g phentolamine, 5g vitamin C and 1L DEME culture solution.
所述步骤（3）中，胎盘剪碎至4cm，酶解液室温酶解0.8h。In the step (3), the placenta was shredded to 4 cm, and the enzymatic hydrolysis solution was hydrolyzed at room temperature for 0.8 h.
所述步骤（4）中，将混合液在7℃温度下、1400r/min转速下离心18min，弃上清，加入DEME培养液，得到细胞悬液。In the step (4), the mixture was centrifuged at 7° C. and 1400 r/min for 18 minutes, the supernatant was discarded, and DEME culture medium was added to obtain a cell suspension.
所述步骤（5）中，依次用红细胞裂解液重悬细胞沉淀，室温下裂解残留的红细胞12min，离心后弃上清；用羟乙基淀粉溶液重悬细胞沉淀，室温下静置35min，取上层液体离心后弃去上清；用RPMI-1640培养基重悬细胞沉淀，离心后弃上清，得到造血干细胞。In the step (5), resuspend the cell pellet with erythrocyte lysate in turn, lyse the remaining red blood cells at room temperature for 12 minutes, discard the supernatant after centrifugation; resuspend the cell pellet with hydroxyethyl starch solution, let stand at room temperature for 35 minutes, take After the supernatant was centrifuged, the supernatant was discarded; the cell pellet was resuspended in RPMI-1640 medium, and the supernatant was discarded after centrifugation to obtain hematopoietic stem cells.
所述步骤（6）中，造血干细胞冻存液包括如下重量份的原料：In the step (6), the hematopoietic stem cell cryopreservation solution includes the following raw materials in parts by weight:
二甲基亚砜 12mLDimethyl sulfoxide 12mL
羟乙基淀粉 7gHydroxyethyl starch 7g
细胞培养基 5mLCell culture medium 5mL
人血清白蛋白 7mLHuman Serum Albumin 7mL
人自体血浆 70mL。Human autologous plasma 70mL.
所述步骤（6）中，细胞重悬液的细胞密度为8×106个/mL；程序降温先以1.5℃/min的降温速度降温至-24℃，再以6℃/min的降温速度降温至-79℃。In the step (6), the cell density of the cell resuspension is 8×10 6 cells/mL; the programmed cooling first cools down to -24°C at a cooling rate of 1.5°C/min, and then cools at a cooling rate of 6°C/min Cool down to -79°C.
实施例3Example 3
一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：A method for isolating and extracting hematopoietic stem cells from placenta, comprising the steps of:
（1）胎盘预处理：在无菌手术室环境下采集正常顺产或剖腹产者的健康胎盘，对采集的胎盘进行病原微生物检测，然后用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；(1) Placenta pretreatment: collect healthy placentas from normal vaginal delivery or cesarean section in a sterile operating room environment, conduct pathogenic microorganism detection on the collected placenta, and then clean the placenta with cleaning solution in the early stage, and collect the treated liquid A ;
（2）胎盘灌洗：用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；(2) Placental perfusion: Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the liquid B after perfusion;
（3）胎盘酶解：将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；(3) Enzymatic hydrolysis of placenta: Cut the placenta into pieces, digest the placental tissue with enzymatic hydrolysis solution, and collect the liquid C obtained after enzymatic hydrolysis;
（4）混合离心：将上述收集的液体A、液体B与液体C并为混合液，将混合液离心后加入DEME培养液混合均匀，得到细胞悬液，将细胞悬液离心后取细胞沉淀；(4) Mixed centrifugation: Combine the liquid A, liquid B and liquid C collected above to form a mixed solution, centrifuge the mixed solution and add DEME culture medium to mix evenly to obtain a cell suspension, centrifuge the cell suspension and take the cell pellet;
（5）重悬离心：将细胞沉淀经过多次重悬离心处理，获得造血干细胞；(5) Resuspension and centrifugation: the cell pellet was resuspended and centrifuged several times to obtain hematopoietic stem cells;
（6）细胞冻存：将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，得到细胞重悬液，将细胞重悬液分装入冻存袋中，再将冻存袋装入冻存袋盒中，进行程序降温至-80℃后转至-196℃液氮罐冻存。(6) Cell cryopreservation: add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution and mix evenly to obtain a cell resuspension. Divide the cell resuspension into cryopreservation bags, and then put the cryopreservation bags into freezer bags. In the storage bag box, the temperature was lowered to -80°C and then transferred to -196°C liquid nitrogen tank for freezing.
所述步骤（1）中，检测的项目包括肝炎病毒检测、艾滋病毒检测、性病检测、组织配型检测、造血干/祖细胞定性检测。In the step (1), the detection items include hepatitis virus detection, HIV detection, STD detection, tissue matching detection, and qualitative detection of hematopoietic stem/progenitor cells.
所述步骤（1）中，清洗液由青霉素-链霉素双抗与生理盐水按质量比为1:1混合制得，浓度为100mg/L。In the step (1), the cleaning solution is prepared by mixing penicillin-streptomycin double antibody and normal saline at a mass ratio of 1:1, and the concentration is 100 mg/L.
所述步骤（2）中，所述灌洗液由200mg青霉素-链霉素双抗、0.07g酚妥拉明，60mg赖氨酸、2g维生素C、3mg AMD3100、2支肝素钠注射液（1mL：5mg）、2支硝酸甘油注射液（2mL：12500U）、50mL质量分数为10%的PBS缓冲液和1L DEME培养液制得。In the step (2), the lavage solution is composed of 200mg penicillin-streptomycin double antibody, 0.07g phentolamine, 60mg lysine, 2g vitamin C, 3mg AMD3100, 2 heparin sodium injections (1mL : 5mg), 2 injections of nitroglycerin (2mL: 12500U), 50mL of 10% PBS buffer and 1L of DEME culture solution.
所述步骤（3）中，酶解液中由0.08g胶原酶Ⅷ、0.08g胰酶、3000U肝素钠、0.3g酚妥拉明、6g维生素C和1L DEME培养液制得。In the step (3), the enzymolysis solution is prepared from 0.08g collagenase VIII, 0.08g trypsin, 3000U heparin sodium, 0.3g phentolamine, 6g vitamin C and 1L DEME culture solution.
所述步骤（3）中，胎盘剪碎至4.5cm，酶解液室温酶解1h。In the step (3), the placenta was shredded to 4.5 cm, and the enzymatic hydrolysis solution was hydrolyzed at room temperature for 1 hour.
所述步骤（4）中，将混合液在8℃温度下、1500r/min转速下离心15min，弃上清，加入DEME培养液，得到细胞悬液。In the step (4), the mixture was centrifuged at 8° C. and 1500 r/min for 15 minutes, the supernatant was discarded, and DEME culture medium was added to obtain a cell suspension.
所述步骤（5）中，依次用红细胞裂解液重悬细胞沉淀，室温下裂解残留的红细胞15min，离心后弃上清；用羟乙基淀粉溶液重悬细胞沉淀，室温下静置40min，取上层液体离心后弃去上清；用RPMI-1640培养基重悬细胞沉淀，离心后弃上清，得到造血干细胞。In the step (5), resuspend the cell pellet with erythrocyte lysate in turn, lyse the residual red blood cells at room temperature for 15 minutes, discard the supernatant after centrifugation; resuspend the cell pellet with hydroxyethyl starch solution, let stand at room temperature for 40 minutes, take After the supernatant was centrifuged, the supernatant was discarded; the cell pellet was resuspended in RPMI-1640 medium, and the supernatant was discarded after centrifugation to obtain hematopoietic stem cells.
所述步骤（6）中，造血干细胞冻存液包括如下重量份的原料：In the step (6), the hematopoietic stem cell cryopreservation solution includes the following raw materials in parts by weight:
二甲基亚砜 15mLDimethyl sulfoxide 15mL
羟乙基淀粉 8gHydroxyethyl starch 8g
细胞培养基 6mLCell culture medium 6mL
人血清白蛋白 8mLHuman Serum Albumin 8mL
人自体血浆 80mL。Human autologous plasma 80mL.
所述步骤（6）中，细胞重悬液的细胞密度为1×107个/mL；程序降温先以1.5℃/min的降温速度降温至-25℃，再以6℃/min的降温速度降温至-80℃。In the step (6), the cell density of the cell resuspension is 1×10 7 cells/mL; the programmed cooling first cools down to -25°C at a cooling rate of 1.5°C/min, and then cools at a cooling rate of 6°C/min Cool down to -80°C.
实施例4Example 4
一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：A method for isolating and extracting hematopoietic stem cells from placenta, comprising the steps of:
（1）胎盘预处理：在无菌手术室环境下采集正常顺产或剖腹产者的健康胎盘，对采集的胎盘进行病原微生物检测，然后用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；(1) Placenta pretreatment: collect healthy placentas from normal vaginal delivery or cesarean section in a sterile operating room environment, conduct pathogenic microorganism detection on the collected placenta, and then clean the placenta with cleaning solution in the early stage, and collect the treated liquid A ;
（2）胎盘灌洗：用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；(2) Placental perfusion: Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the liquid B after perfusion;
（3）胎盘酶解：将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；(3) Enzymatic hydrolysis of placenta: Cut the placenta into pieces, digest the placental tissue with enzymatic hydrolysis solution, and collect the liquid C obtained after enzymatic hydrolysis;
（4）混合离心：将上述收集的液体A、液体B与液体C并为混合液，将混合液离心后加入DEME培养液混合均匀，得到细胞悬液，将细胞悬液离心后取细胞沉淀；(4) Mixed centrifugation: Combine the liquid A, liquid B and liquid C collected above to form a mixed solution, centrifuge the mixed solution and add DEME culture medium to mix evenly to obtain a cell suspension, centrifuge the cell suspension and take the cell pellet;
（5）重悬离心：将细胞沉淀经过多次重悬离心处理，获得造血干细胞；(5) Resuspension and centrifugation: the cell pellet was resuspended and centrifuged several times to obtain hematopoietic stem cells;
（6）细胞冻存：将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，得到细胞重悬液，将细胞重悬液分装入冻存袋中，再将冻存袋装入冻存袋盒中，进行程序降温至-81℃后转至-196℃液氮罐冻存。(6) Cell cryopreservation: add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution and mix evenly to obtain a cell resuspension. Divide the cell resuspension into cryopreservation bags, and then put the cryopreservation bags into freezer bags. In the storage bag box, the temperature was lowered to -81°C and then transferred to -196°C liquid nitrogen tank for freezing.
所述步骤（1）中，检测的项目包括肝炎病毒检测、艾滋病毒检测、性病检测、组织配型检测、造血干/祖细胞定性检测。In the step (1), the detection items include hepatitis virus detection, HIV detection, STD detection, tissue matching detection, and qualitative detection of hematopoietic stem/progenitor cells.
所述步骤（1）中，清洗液由青霉素-链霉素双抗与生理盐水按质量比为1:1.1混合制得，浓度为110mg/L。In the step (1), the cleaning solution is prepared by mixing penicillin-streptomycin double antibody and normal saline at a mass ratio of 1:1.1, with a concentration of 110 mg/L.
所述步骤（2）中，所述灌洗液由250mg青霉素-链霉素双抗、0.08g酚妥拉明，70mg赖氨酸、2.5g维生素C、4mg AMD3100、2支肝素钠注射液（1mL：5mg）、2支硝酸甘油注射液（2mL：12500U）、60mL质量分数为11%的PBS缓冲液和1L DEME培养液制得。In the step (2), the lavage solution is composed of 250 mg penicillin-streptomycin double antibody, 0.08 g phentolamine, 70 mg lysine, 2.5 g vitamin C, 4 mg AMD3100, 2 heparin sodium injections ( 1mL: 5mg), 2 injections of nitroglycerin (2mL: 12500U), 60mL of 11% PBS buffer and 1L DEME culture solution.
所述步骤（3）中，酶解液中由0.09g胶原酶Ⅷ、0.09g胰酶、4000U肝素钠、0.4g酚妥拉明、7g维生素C和1L DEME培养液制得。In the step (3), the enzymolysis solution is prepared from 0.09g collagenase VIII, 0.09g trypsin, 4000U heparin sodium, 0.4g phentolamine, 7g vitamin C and 1L DEME culture solution.
所述步骤（3）中，胎盘剪碎至5cm，酶解液室温酶解1.2h。In the step (3), the placenta was shredded to 5 cm, and the enzymatic hydrolysis solution was hydrolyzed at room temperature for 1.2 hours.
所述步骤（4）中，将混合液在9℃温度下、1600r/min转速下离心12min，弃上清，加入DEME培养液，得到细胞悬液。In the step (4), the mixture was centrifuged at 9° C. and 1600 r/min for 12 minutes, the supernatant was discarded, and DEME culture medium was added to obtain a cell suspension.
所述步骤（5）中，依次用红细胞裂解液重悬细胞沉淀，室温下裂解残留的红细胞18min，离心后弃上清；用羟乙基淀粉溶液重悬细胞沉淀，室温下静置45min，取上层液体离心后弃去上清；用RPMI-1640培养基重悬细胞沉淀，离心后弃上清，得到造血干细胞。In the step (5), resuspend the cell pellet with erythrocyte lysate in turn, lyse the remaining red blood cells at room temperature for 18 minutes, discard the supernatant after centrifugation; resuspend the cell pellet with hydroxyethyl starch solution, let stand at room temperature for 45 minutes, take After the supernatant was centrifuged, the supernatant was discarded; the cell pellet was resuspended in RPMI-1640 medium, and the supernatant was discarded after centrifugation to obtain hematopoietic stem cells.
所述步骤（6）中，造血干细胞冻存液包括如下重量份的原料：In the step (6), the hematopoietic stem cell cryopreservation solution includes the following raw materials in parts by weight:
二甲基亚砜 18mLDimethyl sulfoxide 18mL
羟乙基淀粉 9gHydroxyethyl starch 9g
细胞培养基 7mLCell culture medium 7mL
人血清白蛋白 9mLHuman Serum Albumin 9mL
人自体血浆 90mL。Human autologous plasma 90mL.
所述步骤（6）中，细胞重悬液的细胞密度为3×107个/mL；程序降温先以1.5℃/min的降温速度降温至-26℃，再以6℃/min的降温速度降温至-81℃。In the step (6), the cell density of the cell resuspension is 3×10 7 cells/mL; the programmed cooling first cools down to -26°C at a cooling rate of 1.5°C/min, and then cools at a cooling rate of 6°C/min Cool down to -81°C.
实施例5Example 5
一种从胎盘中分离提取造血干细胞的方法，包括如下步骤：A method for isolating and extracting hematopoietic stem cells from placenta, comprising the steps of:
（1）胎盘预处理：在无菌手术室环境下采集正常顺产或剖腹产者的健康胎盘，对采集的胎盘进行病原微生物检测，然后用清洗液对胎盘进行前期清洗处理，收集处理后的液体A；(1) Placenta pretreatment: collect healthy placentas from normal vaginal delivery or cesarean section in a sterile operating room environment, conduct pathogenic microorganism detection on the collected placenta, and then clean the placenta with cleaning solution in the early stage, and collect the treated liquid A ;
（2）胎盘灌洗：用灌洗液分别灌洗胎盘脐静脉、脐动脉内的血液，收集灌洗后的液体B；(2) Placental perfusion: Perfuse the blood in the umbilical vein and umbilical artery of the placenta with perfusate, and collect the liquid B after perfusion;
（3）胎盘酶解：将胎盘剪碎，用酶解液消化胎盘组织，收集酶解后得到的液体C；(3) Enzymatic hydrolysis of placenta: Cut the placenta into pieces, digest the placental tissue with enzymatic hydrolysis solution, and collect the liquid C obtained after enzymatic hydrolysis;
（4）混合离心：将上述收集的液体A、液体B与液体C并为混合液，将混合液离心后加入DEME培养液混合均匀，得到细胞悬液，将细胞悬液离心后取细胞沉淀；(4) Mixed centrifugation: Combine the liquid A, liquid B and liquid C collected above to form a mixed solution, centrifuge the mixed solution and add DEME culture medium to mix evenly to obtain a cell suspension, centrifuge the cell suspension and take the cell pellet;
（5）重悬离心：将细胞沉淀经过多次重悬离心处理，获得造血干细胞；(5) Resuspension and centrifugation: the cell pellet was resuspended and centrifuged several times to obtain hematopoietic stem cells;
（6）细胞冻存：将造血干细胞等体积加入到造血干细胞冻存液中混合均匀，得到细胞重悬液，将细胞重悬液分装入冻存袋中，再将冻存袋装入冻存袋盒中，进行程序降温至-82℃后转至-196℃液氮罐冻存。(6) Cell cryopreservation: add an equal volume of hematopoietic stem cells to the hematopoietic stem cell cryopreservation solution and mix evenly to obtain a cell resuspension. Divide the cell resuspension into cryopreservation bags, and then put the cryopreservation bags into freezer bags. In the storage bag box, the temperature was lowered to -82°C and then transferred to -196°C liquid nitrogen tank for freezing.
所述步骤（1）中，检测的项目包括肝炎病毒检测、艾滋病毒检测、性病检测、组织配型检测、造血干/祖细胞定性检测。In the step (1), the detection items include hepatitis virus detection, HIV detection, STD detection, tissue matching detection, and qualitative detection of hematopoietic stem/progenitor cells.
所述步骤（1）中，清洗液由青霉素-链霉素双抗与生理盐水按质量比为1:1.2混合制得，浓度为120mg/L。In the step (1), the cleaning solution is prepared by mixing penicillin-streptomycin double antibody and normal saline at a mass ratio of 1:1.2, with a concentration of 120 mg/L.
所述步骤（2）中，所述灌洗液由300mg青霉素-链霉素双抗、0.09g酚妥拉明，80mg赖氨酸、3g维生素C、5mg AMD3100、2支肝素钠注射液（1mL：5mg）、2支硝酸甘油注射液（2mL：12500U）、70mL质量分数为8%-12%的PBS缓冲液和1L DEME培养液制得。In the step (2), the lavage solution is composed of 300mg penicillin-streptomycin double antibody, 0.09g phentolamine, 80mg lysine, 3g vitamin C, 5mg AMD3100, 2 heparin sodium injections (1mL : 5mg), 2 nitroglycerin injections (2mL: 12500U), 70mL PBS buffer solution with a mass fraction of 8%-12%, and 1L DEME culture solution.
所述步骤（3）中，酶解液中由0.10g胶原酶Ⅷ、0.10g胰酶、5000U肝素钠、0.5g酚妥拉明、8g维生素C和1L DEME培养液制得。In the step (3), the enzymolysis solution is prepared from 0.10g collagenase VIII, 0.10g trypsin, 5000U heparin sodium, 0.5g phentolamine, 8g vitamin C and 1L DEME culture solution.
所述步骤（3）中，胎盘剪碎至6cm，酶解液室温酶解1.5h。In the step (3), the placenta is shredded to 6 cm, and the enzymatic hydrolysis solution is hydrolyzed at room temperature for 1.5 h.
所述步骤（4）中，将混合液在10℃温度下、1800r/min转速下离心10min，弃上清，加入DEME培养液，得到细胞悬液。In the step (4), the mixture was centrifuged at 10° C. and 1800 r/min for 10 minutes, the supernatant was discarded, and DEME culture solution was added to obtain a cell suspension.
所述步骤（5）中，依次用红细胞裂解液重悬细胞沉淀，室温下裂解残留的红细胞20min，离心后弃上清；用羟乙基淀粉溶液重悬细胞沉淀，室温下静置50min，取上层液体离心后弃去上清；用RPMI-1640培养基重悬细胞沉淀，离心后弃上清，得到造血干细胞。In the step (5), resuspend the cell pellet with erythrocyte lysate in turn, lyse the remaining red blood cells at room temperature for 20 minutes, discard the supernatant after centrifugation; resuspend the cell pellet with hydroxyethyl starch solution, let stand at room temperature for 50 minutes, take After the supernatant was centrifuged, the supernatant was discarded; the cell pellet was resuspended in RPMI-1640 medium, and the supernatant was discarded after centrifugation to obtain hematopoietic stem cells.
所述步骤（6）中，造血干细胞冻存液包括如下重量份的原料：In the step (6), the hematopoietic stem cell cryopreservation solution includes the following raw materials in parts by weight:
二甲基亚砜 20mLDimethyl sulfoxide 20mL
羟乙基淀粉 10gHydroxyethyl starch 10g
细胞培养基 8mLCell culture medium 8mL
人血清白蛋白 10mLHuman Serum Albumin 10mL
人自体血浆 100mL。Human autologous plasma 100mL.
所述步骤（6）中，细胞重悬液的细胞密度为5×107个/mL；程序降温先以2℃/min的降温速度降温至-27℃，再以7℃/min的降温速度降温至-82℃。In the step (6), the cell density of the cell resuspension is 5×10 7 cells/mL; the programmed cooling first cools down to -27°C at a cooling rate of 2°C/min, and then cools at a cooling rate of 7°C/min Cool down to -82°C.
实施例6Example 6
本实施例与上述实施例1的不同之处在于：The difference between this embodiment and the above-mentioned embodiment 1 is:
所述细胞培养基为X-VIVO15培养基。The cell culture medium is X-VIVO15 medium.
所述人自体血浆的制备方法为：将人体外周血置于无菌离心管中，1500rpm转速下离心8min，取上层血浆在56℃温度下灭活32min后，转移至新的离心管中，2500rpm转速下离心15min，吸取上清，即为冻存用人自体血浆。The preparation method of the human autologous plasma is as follows: put human peripheral blood in a sterile centrifuge tube, centrifuge at 1500rpm for 8min, take the upper plasma and inactivate it at 56°C for 32min, then transfer it to a new centrifuge tube at 2500rpm Centrifuge at a rotating speed for 15 minutes, and absorb the supernatant, which is the human autologous plasma for cryopreservation.
所述造血干细胞冻存液还包括糖类1g，所述糖类是由海藻糖、α-1 ,4-葡聚糖、香菇多糖和葡萄糖以重量比1:0.8：1.5：2组成的混合物。The hematopoietic stem cell cryopreservation solution also includes 1 g of sugar, which is a mixture of trehalose, α-1,4-glucan, lentinan and glucose in a weight ratio of 1:0.8:1.5:2.
所述造血干细胞冻存液还包括非必须氨基酸0.2g和维生素C 0.1g。The hematopoietic stem cell cryopreservation solution also includes 0.2 g of non-essential amino acids and 0.1 g of vitamin C.
所述造血干细胞冻存液还包括丙二醇0.2mL和聚乙二醇0.4mL。The hematopoietic stem cell cryopreservation solution also includes 0.2 mL of propylene glycol and 0.4 mL of polyethylene glycol.
所述造血干细胞冻存液还包括重组人白介素4000U。The hematopoietic stem cell cryopreservation solution also includes recombinant human interleukin 4000U.
所述造血干细胞冻存液还包括白酒草皂苷R 0.3g和勃脉力A 0.8g。The hematopoietic stem cell cryopreservation solution also includes 0.3 g of Liquor saponin R and 0.8 g of Bomaili A.
所述造血干细胞冻存液还包括青霉素-链霉素双抗溶液0.1mL和氯化钠0.4g。The hematopoietic stem cell cryopreservation solution also includes 0.1 mL of penicillin-streptomycin double antibody solution and 0.4 g of sodium chloride.
实施例7Example 7
本实施例与上述实施例2的不同之处在于：The difference between this embodiment and the above-mentioned embodiment 2 is:
所述细胞培养基为AIM-V培养基。The cell culture medium is AIM-V medium.
所述人自体血浆的制备方法为：将人体外周血置于无菌离心管中，1600rpm转速下离心7min，取上层血浆在57℃温度下灭活31min后，转移至新的离心管中，2800rpm转速下离心22min，吸取上清，即为冻存用人自体血浆。The preparation method of the human autologous plasma is as follows: human peripheral blood is placed in a sterile centrifuge tube, centrifuged at 1600rpm for 7min, the upper layer of plasma is inactivated at 57°C for 31min, and then transferred to a new centrifuge tube at 2800rpm Centrifuge at a rotational speed for 22 minutes, and absorb the supernatant, which is the human autologous plasma for cryopreservation.
所述造血干细胞冻存液还包括糖类1.5g，所述糖类是由海藻糖、α-1 ,4-葡聚糖、香菇多糖和葡萄糖以重量比1:0.9：1.8：2.5组成的混合物。The hematopoietic stem cell cryopreservation solution also includes 1.5 g of carbohydrates, which are a mixture of trehalose, α-1,4-glucan, lentinan and glucose in a weight ratio of 1:0.9:1.8:2.5 .
所述造血干细胞冻存液还包括非必须氨基酸0.3g和维生素C 0.2g。The hematopoietic stem cell cryopreservation solution also includes 0.3 g of non-essential amino acids and 0.2 g of vitamin C.
所述造血干细胞冻存液还包括丙二醇0.2-0.6mL和聚乙二醇0.4-0.8mL。The hematopoietic stem cell cryopreservation solution also includes 0.2-0.6 mL of propylene glycol and 0.4-0.8 mL of polyethylene glycol.
所述造血干细胞冻存液还包括重组人白介素5000U。The hematopoietic stem cell cryopreservation solution also includes 5000 U of recombinant human interleukin.
所述造血干细胞冻存液还包括白酒草皂苷R 0.4g和勃脉力A 0.9g。The hematopoietic stem cell cryopreservation solution also includes 0.4 g of Liquor saponin R and 0.9 g of Bomaili A.
所述造血干细胞冻存液还包括青霉素-链霉素双抗溶液0.2mL和氯化钠0.5g。The hematopoietic stem cell cryopreservation solution also includes 0.2 mL of penicillin-streptomycin double antibody solution and 0.5 g of sodium chloride.
实施例8Example 8
本实施例与上述实施例3的不同之处在于：The difference between this embodiment and the above-mentioned embodiment 3 is:
所述细胞培养基为DMEM/F12培养基。The cell culture medium is DMEM/F12 medium.
所述人自体血浆的制备方法为：将人体外周血置于无菌离心管中，1800rpm转速下离心6min，取上层血浆在58℃温度下灭活30min后，转移至新的离心管中，3000rpm转速下离心20min，吸取上清，即为冻存用人自体血浆。The preparation method of the human autologous plasma is as follows: human peripheral blood is placed in a sterile centrifuge tube, centrifuged at 1800rpm for 6min, the upper layer of plasma is inactivated at 58°C for 30min, and then transferred to a new centrifuge tube at 3000rpm Centrifuge at a rotating speed for 20 minutes, and absorb the supernatant, which is the human autologous plasma for cryopreservation.
所述造血干细胞冻存液还包括糖类2g，所述糖类是由海藻糖、α-1 ,4-葡聚糖、香菇多糖和葡萄糖以重量比1:1：2：3组成的混合物。The hematopoietic stem cell cryopreservation solution also includes 2 g of carbohydrates, which are a mixture of trehalose, α-1,4-glucan, lentinan and glucose in a weight ratio of 1:1:2:3.
所述造血干细胞冻存液还包括非必须氨基酸0.4g和维生素C 0.3g。The hematopoietic stem cell cryopreservation solution also includes 0.4 g of non-essential amino acids and 0.3 g of vitamin C.
所述造血干细胞冻存液还包括丙二醇0.4mL和聚乙二醇0.6mL。The hematopoietic stem cell cryopreservation solution also includes 0.4 mL of propylene glycol and 0.6 mL of polyethylene glycol.
所述造血干细胞冻存液还包括重组人白介素6000U。The hematopoietic stem cell cryopreservation solution also includes recombinant human interleukin 6000U.
所述造血干细胞冻存液还包括白酒草皂苷R 0.5g和勃脉力A 1g。The hematopoietic stem cell cryopreservation solution also includes 0.5 g of Liquor saponin R and 1 g of Bomaili A.
所述造血干细胞冻存液还包括青霉素-链霉素双抗溶液0.3mL和氯化钠0.6g。The hematopoietic stem cell cryopreservation solution also includes 0.3 mL of penicillin-streptomycin double antibody solution and 0.6 g of sodium chloride.
实施例9Example 9
本实施例与上述实施例4的不同之处在于：The difference between this embodiment and the above-mentioned embodiment 4 is:
所述细胞培养基为RPMI-1640培养基、GT-T551培养基或GT-T561培养基。The cell culture medium is RPMI-1640 medium, GT-T551 medium or GT-T561 medium.
所述人自体血浆的制备方法为：将人体外周血置于无菌离心管中，1900rpm转速下离心5min，取上层血浆在59℃温度下灭活29min后，转移至新的离心管中，3200rpm转速下离心18min，吸取上清，即为冻存用人自体血浆。The preparation method of the human autologous plasma is as follows: human peripheral blood is placed in a sterile centrifuge tube, centrifuged at 1900rpm for 5min, the upper layer of plasma is inactivated at 59°C for 29min, and then transferred to a new centrifuge tube at 3200rpm Centrifuge at a rotational speed for 18 minutes, and absorb the supernatant, which is human autologous plasma for cryopreservation.
所述造血干细胞冻存液还包括糖类2.5g，所述糖类是由海藻糖、α-1 ,4-葡聚糖、香菇多糖和葡萄糖以重量比1:1.1：2.2：3.5组成的混合物。The hematopoietic stem cell cryopreservation solution also includes 2.5 g of carbohydrates, which are a mixture of trehalose, α-1,4-glucan, lentinan and glucose in a weight ratio of 1:1.1:2.2:3.5 .
所述造血干细胞冻存液还包括非必须氨基酸0.4g和维生素C 0.3g。The hematopoietic stem cell cryopreservation solution also includes 0.4 g of non-essential amino acids and 0.3 g of vitamin C.
所述造血干细胞冻存液还包括丙二醇0.4mL和聚乙二醇0.6mL。The hematopoietic stem cell cryopreservation solution also includes 0.4 mL of propylene glycol and 0.6 mL of polyethylene glycol.
所述造血干细胞冻存液还包括重组人白介素6000U。The hematopoietic stem cell cryopreservation solution also includes recombinant human interleukin 6000U.
所述造血干细胞冻存液还包括白酒草皂苷R 0.3-0.7g和勃脉力A 0.8-1.2g。The hematopoietic stem cell cryopreservation solution also includes 0.3-0.7 g of Liquor saponin R and 0.8-1.2 g of Bomaili A.
所述造血干细胞冻存液还包括青霉素-链霉素双抗溶液0.3mL和氯化钠0.6g。The hematopoietic stem cell cryopreservation solution also includes 0.3 mL of penicillin-streptomycin double antibody solution and 0.6 g of sodium chloride.
实施例10Example 10
本实施例与上述实施例5的不同之处在于：The difference between this embodiment and the above-mentioned embodiment 5 is:
所述细胞培养基为Stemspan培养基。The cell culture medium is Stemspan medium.
所述人自体血浆的制备方法为：将人体外周血置于无菌离心管中， 2000rpm转速下离心4min，取上层血浆在60℃温度下灭活28min后，转移至新的离心管中，3500rpm转速下离心15min，吸取上清，即为冻存用人自体血浆。The preparation method of the human autologous plasma is as follows: human peripheral blood is placed in a sterile centrifuge tube, centrifuged at 2000rpm for 4min, the upper layer of plasma is inactivated at 60°C for 28min, and then transferred to a new centrifuge tube at 3500rpm Centrifuge at a rotating speed for 15 minutes, and absorb the supernatant, which is the human autologous plasma for cryopreservation.
所述造血干细胞冻存液还包括糖类3g，所述糖类是由海藻糖、α-1 ,4-葡聚糖、香菇多糖和葡萄糖以重量比1:1.2：2.5：4组成的混合物。The hematopoietic stem cell cryopreservation solution also includes 3 g of carbohydrates, which are a mixture of trehalose, α-1,4-glucan, lentinan and glucose in a weight ratio of 1:1.2:2.5:4.
所述造血干细胞冻存液还包括非必须氨基酸0.6g和维生素C 0.5g。The hematopoietic stem cell cryopreservation solution also includes 0.6 g of non-essential amino acids and 0.5 g of vitamin C.
所述造血干细胞冻存液还包括丙二醇0.6mL和聚乙二醇0.8mL。The hematopoietic stem cell cryopreservation solution also includes 0.6 mL of propylene glycol and 0.8 mL of polyethylene glycol.
所述造血干细胞冻存液还包括重组人白介素8000U和肝素钠5000U。The hematopoietic stem cell cryopreservation solution also includes 8000 U of recombinant human interleukin and 5000 U of heparin sodium.
所述造血干细胞冻存液还包括白酒草皂苷R 0.7g和勃脉力A 1.2g。The hematopoietic stem cell cryopreservation solution also includes 0.7g of Liquor saponin R and 1.2g of Bomaili A.
所述造血干细胞冻存液还包括青霉素-链霉素双抗溶液0.5mL和氯化钠0.8g。The hematopoietic stem cell cryopreservation solution also includes 0.5 mL of penicillin-streptomycin double antibody solution and 0.8 g of sodium chloride.
将实施例1-5的方法从胎盘中分离提取造血干细胞，经1个月、3个月、6个月、12个月冻存后依次取出复苏，检测造血干细胞存活率，实验结果如下表所示：The hematopoietic stem cells were isolated and extracted from the placenta according to the method of Examples 1-5, and after 1 month, 3 months, 6 months, and 12 months of freezing, they were sequentially taken out and resuscitated, and the survival rate of hematopoietic stem cells was detected. The experimental results are shown in the following table Show:
从上表可以看出，本发明的方法节省了制备时间，能够有效提高造血干细胞分离率和细胞冻存复苏后的存活率；操作方便，效率高，大大缩短的从制备到应用的周期；且造血干细胞损伤小，数量多，纯度高，活性高，可以应用于自体或者异体的造血干细胞的移植，并且有助于受损组织的修复。As can be seen from the above table, the method of the present invention saves preparation time, can effectively improve the isolation rate of hematopoietic stem cells and the survival rate after cell cryopreservation and resuscitation; the operation is convenient, the efficiency is high, and the cycle from preparation to application is greatly shortened; and Hematopoietic stem cells are small in damage, large in number, high in purity, and high in activity, and can be applied to autologous or allogeneic hematopoietic stem cell transplantation, and help repair damaged tissues.
上述实施例为本发明较佳的实现方案，除此之外，本发明还可以其它方式实现，在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。The above-mentioned embodiments are preferred implementation solutions of the present invention. In addition, the present invention can also be realized in other ways, and any obvious replacements are within the protection scope of the present invention without departing from the concept of the present invention.
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| CN108148806A (en) * | 2018-02-02 | 2018-06-12 | 中国人民解放军军事科学院军事医学研究院 | A kind of fast separating process of placental hematopoietic stem cell |
| CN109337871A (en) * | 2018-11-05 | 2019-02-15 | 潍坊市康华生物技术有限公司 | A kind of preparation method of placental hematopoietic stem cells |
| CN109652372A (en) * | 2019-01-09 | 2019-04-19 | 陕西九州细胞基因工程有限公司 | A kind of quick separating of human placenta source candidate stem cell, preparation method |
| CN110734892A (en) * | 2019-11-26 | 2020-01-31 | 深圳科学之门生物工程有限公司 | method for extracting hematopoietic stem cells from placenta hominis |
| CN115537391A (en) * | 2022-11-11 | 2022-12-30 | 朗姿赛尔生物科技（广州）有限公司 | A method for extracting and preserving hematopoietic stem cells |
| CN117778314A (en) * | 2023-12-28 | 2024-03-29 | 广东唯泰生物科技有限公司 | A method for isolating nucleated cells derived from placenta |
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